Reliable ChIP-seq results with the Diagenode True MicroChIP Kit andMicroPlex Library Preparation Kit on only 10,000 cells
Céline Sabatel, Irina Panteleeva, Miklos Laczik, Geoffrey Berguet, Ignacio Mazon*, Sharon Squazzo*, Hélène Pendeville and Dominique Poncelet
Diagenode sa, CHU, Tour GIGA B34, 3ème étage 1 Avenue de l’Hôpital, 4000 Liège, Sart-Tilman, Belgium I *Diagenode inc., 400 Morris Avenue, Suite 101, Denville, NJ 07834, USA

MicroPlex library preparation kit protocol overview

Automated ChIP

The Diagenode True MicroChIP Kit has been developed with both an optimized protocol and reagents toenable successful ChIP on 10,000 cells. Moreover, the True MicroChIP kit protocol has been thoroughly
optimized by Diagenode for ChIP followed by high-throughput sequencing on an Illumina® sequencer.
In addition, to enable sequencing on the low amounts of DNA recovered after ChIP on 10,000 cells,
Diagenode has developed a library preparation protocol for limited quantities of DNA. The MicroPlex
Library Preparation Kit requires only picogram amounts of ChIP’d DNA inputs for library preparationcompatible with the Illumina® platforms.

1. 6X faster than TruSeq

Add Pre-Mix

Add Pre-Mix

 3 incubations in a single tube
 No intermediate purifications
 No transfers and no columns
2 Hours
 Manual throughput up to 192 samples per day with one operator
dsDNA template

Cleavable
Replication
StopxxIn this poster, we demontrated the successful use of the Diagenode True MicroChIP Kit in combination
with the MicroPlex Library Preparation Kit for ChIP-seq on the Illumina® platform.

+Blunt-end Ligation
Nick

NickIntroduction

Stem-loop
AdaptorExtension and CleavageEIF4A2p
GAPDHp
MB2Sat2104. Greater coverage of complex
sequences05. Automatable, indexed

H3K4me3IgG

Manual ChIP
4050 pg to 50 ng of fragmented ds DNA are converted intosequencing-ready libraries for Illumina® NGS platforms
using a fast, simple and sensitive 3-step protocol. After
amplification, libraries are purified with AMPure XP
beads. The purified libraries are ready for sequencing
on an Illumina® platform. Indexing reagents included in
the kit allow the multiplexing of 12 samples in a single
sequencing lane.

CleavableReplicationstop

Addition of Adaptors+3. Simple preps

Figure 2. MicroPlex library preparation workflow.

DNA Repair
Stem-loopAdaptor

2. 100X more sensitive%input

Mix Reagent
and sampleNGS
Template%input

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) has become thegold standard for whole-genome mapping of protein-DNA interactions. However, conventional ChIP
protocols require abundant amounts of starting material (at least hundreds of thousands of cells per
immunoprecipitation) limiting the application for the ChIP technology to few cell samples.

20TMEIF4A2p
GAPDHp
MB2Sat2200H3K4me3

4. Library preparation on low amount of ChIP’d DNA with no pre-amplification step using the MicroPlex kit
Figure 6. Library generation with theMicroPlex kit.

A.

ChIP assays were performed on
10,000 and 100,000 HeLa cells with
Diagenode H3K4me3 antibody (0.25 µg/reaction). Libraries were made withthe MicroPlex Library Preparation
Kit. The generated libraries were thenanalysed on an Illumina® HiSeq2000.
Cluster generation and sequencing
were performed according to themanufacturer’s instructions.

BarcodeThe True MicroChIP Kit has been developed specifically for ChIP with limited cell numbers by optimizing
several parameters; such as:

Primer BResults• Chromatin shearing• Antibody concentration• Use of different carriers• Stringency of washes

1. Efficient and easy chromatin shearing using Bioruptor® Pico and True MicroChIP kit Shearing Buffer
Figure 3. HeLa cells were fixed with 1% formaldehyde (for 10 minutes at RT). Cell lysis was performed using
the Lysis Buffer of the Diagenode True MicroChIP kit. Samples corresponding to 10 000 cells were sheared
during 5 rounds of 5 cycles of 30 seconds “ON” / 30 seconds “OFF” with the Bioruptor ® Pico. Samples werevortexed before and after performing 5 sonication cycles, followed by a short centrifugation at 4°C. 10 μl ofDNA (equivalent to 60,000 cells) were analysed on a 1.5% agarose gel.

The MicroPlex Library Preparation Kit is specifically designed for library preparation. It requires only
3 steps with a single purification. The kit requires only picogram amounts of DNA as starting material,
allowing the sequencing of the low DNA amounts recovered after ChIP on low cell numbers.

B.

Methods

98%

ChIP-seq workflow

RepairRepairClean-up

Ligateantibodies
ChIPH3K4me3
H3K4me3
ChIP
14
14

A-TailLigateClean-up
Gel purify
Clean-up

NGS-READY DNA< 3 hours
1 tube

(3)

b.

a.

(4)
b.

c.

(5)

c.

d.

(6)

1414

d.

1212
b.

Figure 1

c.

d.
1010

%input%input

a.

8 800

8 8

True MicroChIP procedure: 1. Cell fixation and DNA-protein cross-linking 2. Cell lysis and6 6 chromatin shearing using the®
4 4Bioruptor Pico 3. Binding of the antibodies to the chromatin.
Addition of beads. 4. Magnetic4immunoprecipitation
5. Washes
4of immune complexes 6. DNA purification and recovery of ChIP’d DNA
ChIP-sequencing procedure: Comparison of the library preparation procedure using the MicroPlex kit and the TruSeq kit
0 00 0®
from Illumina®. The MicroPlex kit uses a 3-step protocol witha
singlepurification
attheendof theprocedure.IgGThe
H3K4me3
H3K4me3
IgG
IgG
H3K27ac
H3K27acIgG Illumina
workflow for library preparation is composed of 5 steps with several purification procedures.

www.diagenode.com

2200

IgG
IgG

H3K27ac
H3K27ac

IgG
IgG

ChIP
ChIPH3K27me3
H3K27me3
1818

Sat2
Sat2ZnF510
ZnF510
cfos
cfos
GAPDH
GAPDH
TSS
TSS

2020

1010

2 2

442525

6 6

2 2

66

H3K4me3
H3K4me3

1515

Myt1
Myt1
TSH2B
TSH2B
GAPDHpGAPDHp
EIF4A2p
EIF4A2p

1616
1414
1212

6 6
4 4

5 5

2 2

TM

0 00 0H3K9me3
H3K9me3

IgG
IgG

25252020

ChIPH3K27me3
H3K27me3
ChIP
1818

Sat2
Sat2ZnF510
ZnF510cfos
cfosGAPDHTSS
TSS
GAPDH

16161414Figure 4. ChIP efficiency on 10,000 cells.

00

The qPCR was
performedIgG
with primers for two
H3K9me3IgG
H3K9me3
positive loci and two negative loci for each ChIP
assay. Figures show the recovery, expressed
as a percentage of input (the relative amountof immunoprecipitated DNA compared to input
DNA after qPCR analysis).

Myt1
Myt1
TSH2B
TSH2B
GAPDHpGAPDHp
EIF4A2p
EIF4A2p2%121200Matched by Broad
Institute dataset
Unmatched by Broad
Institute dataset

A. The 36 bp tags were mapped to thehuman genome with the ELAND aligner.
During the subsequent peak calling by
SICER the enrichments from low cellnumbers could be identified with asmuch confidence as from millions ofcells.
B: The datasets were analyzed andcompared with each other and to thereference data generated by the Broad
Institute. We proved that our low cellsamples are consistent and have very
high similarity, and even the 30 pg
sample fulfils the Encode criteria (min.
80% of the top 40% of the peaks shouldoverlap.)

Conclusions
True MicroChIP Kit:
• The first and unique ChIP kit for very low starting cell numbers• Optimizedfor an automated format
H3K27me3
IgG
H3K27me3
IgG• Fully validated using ChIP-qPCR on multiple key epigenetic marks• ChIP-seq validation using ChIP’d DNA from as low as 10,000 cells with the MicroPlex kit without preamplification
MicroPlex Library Preparation Kit:
• Fast and simplified protocol for NGS library preparation• Efficient library preparation on picogram amount of DNA without pre-amplification• Multiplexing capacity of up to 12 samples using standard Illumina® index tags
• Compatible with all Illumina® sequencing platforms

1010
8 81010ChIPH3K9me3
H3K9me3
ChIP1515
ChIP assays
were performed on 10,000 Hela 1010
cells with several Diagenode antibodies: 88
1010H3K4me3
antibody (0.25 µg/reaction), H3K27ac 66(0.1µg/reaction), H3K9me3 (0.5 µg/reaction) and 4455 (0.25 µg/reaction). Identical quantity
H3K27me3
22of IgG was used as a control.

88

ChIP
ChIPH3K9me3
H3K9me3• 1 μg DNA input recommended• 16
13 user-supplied reagent
16
EIF4A2pEIF4A2p• > 50 pipetting steps
ACTBp
ACTBp
• 148 sample transfers Myt1
14Myt1• 5 purifications
TSH2B
TSH2B
12
• 12
1 clean-up at end

EIF4A2
EIF4A2
prom
prom
GAPDH
GAPDH
prom
prom
MBMB
ex2ex2
Sat2
Sat2

10
1022

NGS-READY DNA
11hours
ChIP
ChIP
H3K27ac
H3K27ac%input%inputa.

121244Amplify
Clean-up• 50 pg - 50 ng DNA input recommended• 1 user-supplied reagent• 22 pipetting steps• No sample transfers and no columns• 1 clean-up at end
ChIP
ChIPH3K4me3
H3K4me3• 192 sample throughput per day per tech

88

EIF4A2p
EIF4A2p
ACTBp
ACTBp
Myt1
Myt1
TSH2B
TSH2B

141466%input%input(2)1010%input%input

Amplify
Clean-up

1616

EIF4A2prom
prom
EIF4A2GAPDH
prom
GAPDH prom
MBex2
MB ex2
Sat2
Sat21212(1)ChIPH3K27ac
H3K27ac
ChIP%input%input

Illumina® TruSeqTM DNA LS Prep Kit%input%input

Bioruptor ® PicoMicroPlex%input%input

iDeal ChIP-seq kit

2. Proven and reliable ChIP results with the True MicroChIP Kit using Diagenode’s Premium

ChIP-seq%input%input

True MicroChIP

ChIP assays were performed on 10,000
Hela cells with the Diagenode antibody
H3K4me3 (0.25 µg/reaction) on the IPStar Compact. 0.25 µg of IgG was used
as a control. The qPCR was performed
with primers for the positive loci EIF4A2
promoter and GAPDH TSS and thenegative loci Myoglobin exon 2 and Sat2.
Figure shows the recovery, expressed
as a percent of input (the relative amountof immunoprecipitated DNA compared
to input DNA after qPCR analysis).

IgG

High Fidelity AmplificationPrimer A

Figure 5. Automation of ChIP assay on
10,000 cells.

H3K27me3
H3K27me3

IgG
IgG

MicroPlex Library Preparation kit x12 contains ThruPLEX technology developed and manufactured by Rubicon Genomics, Inc.,Ann Arbor,Michigan, USA and covered by US Patent 7,803,550; EP1924704; and US and international patents pending.
The development of the True MicroChIP kit has partly been funded by FP-7 collaborative project (N°221952)

For more information, please contact: for Europe, info@diagenode.com & for the US and Canada, contact info.na@diagenode.com

PO-µChIP-µPLEX-A3-V1_21_01_13

Abstract

3. True MicroChIP kit compatible with Automated System IP-Star® Compact